High Fat Diet Rapidly Suppresses B Lymphopoiesis byDisrupting the Supportive Capacity of the Bone MarrowNicheBenjamin J. Adler, Danielle E. Green, Gabriel M. Pagnotti, M. Ete Chan, Clinton T. Rubin*

Department of Biomedical Engineering, Stony Brook University, Stony Brook, New York, United States of America

Abstract

The bone marrow (BM) niche is the primary site of hematopoiesis, and cues from this microenvironment are critical tomaintain hematopoiesis. Obesity increases lifetime susceptibility to a host of chronic diseases, and has been linked todefective leukogenesis. The pressures obesity exerts on hematopoietic tissues led us to study the effects of a high fat diet(HFD: 60% Kcal from fat) on B cell development in BM. Seven week old male C57Bl/6J mice were fed either a high fat (HFD)or regular chow (RD) diet for periods of 2 days, 1 week and 6 weeks. B-cell populations (B220+) were not altered after 2 d ofHFD, within 1 w B-cell proportions were reduced by 210%, and by 6 w by 225% as compared to RD (p,0.05). BM RNA wasextracted to track the expression of B-cell development markers Il-7, Ebf-1 and Pax-5. At 2 d, the expression of Il-7 and Ebf-1were reduced by 220% (p = 0.08) and 211% (p = 0.06) whereas Pax-5 was not significantly impacted. At one week, however,the expressions of Il-7, Ebf-1, and Pax-5 in HFD mice fell by -19%, 220% and 216%, and by six weeks were further reducedto 223%, 229% and 234% as compared to RD (p,0.05 for all), a suppression paralleled by a +363% increase in adiposeencroachment within the marrow space (p,0.01). Il-7 is a critical factor in the early B-cell lineage which is secreted bysupportive cells in the BM niche, and is necessary for B-cell commitment. These data indicate that BM Il-7 expression, and byextension B-cell differentiation, are rapidly impaired by HFD. The trend towards suppressed expression of Il-7 following only2 d of HFD demonstrates how susceptible the BM niche, and the cells which rely on it, are to diet, which ultimately couldcontribute to disease susceptibility in metabolic disorders such as obesity.

Citation: Adler BJ, Green DE, Pagnotti GM, Chan ME, Rubin CT (2014) High Fat Diet Rapidly Suppresses B Lymphopoiesis by Disrupting the Supportive Capacity ofthe Bone Marrow Niche. PLoS ONE 9(3): e90639. doi:10.1371/journal.pone.0090639

Editor: Andreas Zirlik, University Heart Center Freiburg, Germany

Received May 22, 2013; Accepted February 3, 2014; Published March 4, 2014

Copyright: � 2014 Adler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Funding provided by National Institutes of Health (NIH) AR 43498 and EB 14351. The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: Clinton.Rubin@stonybrook.edu

Introduction

Hematopoietic stem cells (HSC), which reside in the bone

marrow, are the source for all red blood cells, platelets and

leukocytes. Leukocytes, generally described as arising from two

distinct hematopoietic lineages, myeloid or lymphoid, orchestrate

the body’s defenses to disease and infection. HSCs ensure stable

hematopoietic activity as well as the ability to respond to acute

challenges to the organism [1,2]. Hematopoietic insults, such as

bleeding or irradiation, are followed by the rapid expansion of

progenitor cells to repair damaged populations, replace blood, and

provide the appropriate immunologic response to combat

infection [3]. Indeed, the rapid expansion of progenitors following

hematopoietic injury represents a critical step in restoring a fully

functioning immune system, by reestablishing depleted or unbal-

anced cell compartments.

To a large degree, hematopoietic capacity and responsiveness is

dependent on the status of the bone marrow microenvironment,

which provides metabolic, physiologic and regulatory cues to the

HSC, balancing quiescence and differentiation [4]. HSCs in the

bone marrow microniche are co-localized, interact with, and

influenced by a host of neighboring cells, including osteoblasts,

osteoclasts, adipocytes and bone marrow stromal cells (BMSC), as

well as adjacent tissues such as vasculature and bone trabecular

and cortical surfaces [2,5,6]. In addition to primitive HSCs,

various hematopoietic progeny which develop in the BM are also

influenced in their maturation by environmental, physical and

chemical signals from the niche. B-cell development in particular

requires signals derived from bone marrow stromal cells and

osteoblasts, among others, such as Il-7 which is critical for B-cell

lineage commitment [7,8]. Given the sensitivity of these immune

cells to environmental cues, it should not be surprising that factors

which compromise bone marrow composition may disrupt normal

production of leukocyte populations.

Obesity increases the lifetime risk to a range of chronic illnesses

[9], susceptibility fostered by a depressed and unresponsive

immune system [10]. Considering that, over time, obesity causes

an age-like shift of the bone marrow towards adiposity, it is

possible that this encroaching fat phenotype influences the fate

selection of progenitor populations and disrupts their viability [11],

ultimately impairing hematopoietic reparative and regenerative

potential [12,13]. Various reports have already indicated that the

obesity caused by long term exposure to high fat diet (HFD) will

disrupt the normal production of leukocytes [14,15,16]. Never-

theless, it is not yet known how quickly this might occur, and if the

diet itself contributes to the disruption of leukogenesis, or if it is

‘simply’ a byproduct of the increased adipose burden. Considering

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the critical role the niche plays in leukogenesis and the

vulnerability of the marrow microenvironment to obesity, we

hypothesize that obesity induced leukocyte defects originate very

early in the process, with changes to the chemical and

morphologic state of the bone marrow arising well before the

obese phenotype is evident. The study reported here is designed to

establish the rapidity in which a high fat diet disrupts the chemical

and physical state of the bone marrow niche, and the degree to

which this impacts the development and balance of immune cell

populations.

Materials and Methods

Animals and Experimental DesignAll animal protocols were reviewed and approved by Stony

Brook University’s IACUC. Male, C57BL/6J mice were obtained

from The Jackson Laboratory (Bar Harbor, ME) at five weeks of

age and housed in a dedicated animal facility. At seven weeks of

age, weight matching was used to evenly distribute animals into

either Regular Diet (RD), or High Fat (HF) groups. At baseline,

mice were fed ad libitum either a standard rodent diet chow (RDC;

14% kcal from fat, LabDiet, ProLab RMH 3000), or a high fat

diet (HFD; 60% kcal from fat, TestDiet, ‘‘Van Heek’’ 58Y1).

Three time points were examined in this study with a cohort of

each group being sacrificed at 2 days, 1 week, and 6 weeks after

the study start. In a separate study to determine the impact of

expanding adipose tissue on circulating immune populations, we

investigated changes to leukocytes residing in adipose tissues

following 3 weeks of HFD. All assays had a sample size of

n = 8–10.

mCT Imaging for the Assessment of Abdominal AdiposityAt the end of the six week protocol, animals were anesthetized

with isoflurane inhalation and scanned in vivo using high-resolution

micro-computed tomography mCT (VivaCT 40, Scanco, Bassers-

dorf, Switzerland). An abdominal region of interest spanning the

L1–L5 vertebral levels was scanned at an isotropic resolution of

76 mm (45 kV, 133 mA, 300 ms integration time) [17]. A custom-

designed image analysis program was used to segment adipose

from other tissues, as well as delineate the visceral and

subcutaneous adipose compartments [18].

Flow Cytometric Characterization of HematopoiesisAt sacrifice, animals were anesthetized with isoflurane inhala-

tion and peripheral blood was drawn via cardiac puncture. After

euthanasia, bone marrow was extracted from the left femur and

tibia. Following erythrocyte lysis (1X Pharmlyse, BD Biosciences,

San Jose, CA), cellularity was determined using an automated

handheld cytometer (Scepter, Millipore, Billerica, MA). Single-cell

suspensions containing 26106 cells were stained to identify

populations enriched for HSCs in bone marrow, hematopoietic

progenitors in bone marrow, and leukocytes including B, T, and

myeloid lineage cells in bone marrow and peripheral blood.

Hematopoietic progenitors were identified based on the

expression of the canonical HSC phenotype of c-KitHi-LinLow-

Sca-1Hi (KLS). Cells expressing KLS in conjunction with the side

population phenotype (SP-KLS) were considered to be HSCs [19].

Side population staining was performed according to previous

protocols using a violet analogue for the classic Hoechst 33342 dye

(Vybrant DyeCycle Violet, Invitrogen, Carlsbad, CA: V35003)

[20]. SP-KLS cells are enriched for HSC activity in comparison to

KLS cells which, although enriched for HSCs themselves, contain

a significant proportion of progenitors [21].

Circulating (peripheral blood) and bone marrow resident

leukocytes were stained with a protocol to identify B, T, and

myeloid lineage cells on a single plot [21]. T-cells were stained

with CD4 and CD8, and myeloid cells were stained with Mac-1

and Gr-1. B-cells were identified based on their expression of

B220. It is important to note that this analysis did not characterize

all hematopoietic lineages as, for example, it did not include either

megakaryocytes or erythroblasts. Light scatter was used in all cases

to exclude debris from the analysis. All antibodies were purchased

from BD Biosciences. We report both the phenotypic proportion

of a measured population, and an extrapolation of that popula-

tion’s total size.

Characterization of BM Differentiation via RT-PCRAt sacrifice, whole bone marrow was extracted from the right

femur and stored in RNAlater (Ambion, Life Technologies, Grand

Island, NY). In order to extract RNA, aliquots of each sample

were selected and the RNAlater was carefully removed by

centrifugation. Cells were lysed using TRIzol (Invitrogen, Life

Technologies), and chloroform separation was used to isolate RNA

in an aqueous phase. RNA was purified using a Qiagen RNeasy

kit per manufacturer’s instructions, including digestion of

contaminating DNA with DNase (Germantown, MD). The

concentration and quality of each RNA sample was measured

with a Nanodrop ND-1000 spectrophotometer (Thermo Scientific,

Wilmington, DE). All reactions were performed using a StepO-

nePlus RT-PCR system (Applied Biosystems, Life Technologies,

Carlsbad, CA). cDNAs were transcribed from each sample using

Applied Biosystem’s High Capacity cDNA Reverse Transcription

kit (#4368814), and diluted down to a concentration of 1.5 ng/ul,

based on the concentrations measured by the nanodrop device,

and assuming a 1:1 conversion ratio. Taqman gene expression

assays from Applied Biosystems were used to messure the

expression of Il-7 (Mm01295803_m1), Ebf-1 (Mm00395519

_m1), Pax-5 (Mm00435501_m1), Runx1 (Mm01213405_m1),

Crebbp (Mm01342452_m1), CXCL-12 (Mm00445553_m1), and

Angiopoietin-1 (Mm00456503_m1) with mouse Actin beta

(4352341E) used as a housekeeping gene. Fold change was

calculated using the DDCT method.

Characterization of Bone Marrow AdiposityAfter six weeks of protocol, right tibiae were fixed in neutral

buffered formalin for 24 hours and then stored in 70% ethanol.

Bones were dehydrated with increasing concentrations of isopro-

pyl alcohol and then embedded in polymethyl methacrylate. Thin

sections (8 mm) were cut and stained for contrast using Wright-

Giemsa stain (n = 6). Adipocyte ghosts were imaged using an

inverted microscope (Zeiss) at 106 magnification. Marrow

adiposity was quantified in the proximal metaphysis using ImageJ

(NIH) to calculate total marrow area relative to the rest of the bone

marrow.

To confirm the results of histology, femoral bone marrow stored

in RNAlater (for PCR, see above) was assayed for triglyceride

content. After homogenization, 1 ml of each sample was mixed in

three volumes of a chloroform/methanol solution at a concentra-

tion of 3:1 chloroform:methanol. After mixing and centrifugation,

a portion (1 ml) of the lipid containing phase was aspirated and

allowed to evaporate overnight. Following resuspension of the lipid

in isopropyl alcohol (150 ml total volume), a modified protocol was

used to determine the triglyceride content of the marrow in

duplicate (30 ml per sample per replicate) (Serum Triglyceride

Determination Kit, Sigma, St. Louis, Mo.).

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Determination of Leukocyte Subsets within AdiposeTissue by Flow Cytometry

In order to determine the impact of expanding adipose tissue on

circulating immune populations, we investigated changes to

leukocytes residing in adipose tissues as caused by obesity (3 W

of HFD). Epidydimal fat collected at sacrifice was stored in

supplemented media on ice until processing (IMDM, 2% FBS, 1%

Pen/Strep, Gibco). For each sample, the left fat pad was incubated

in collagenase Type II for 20 minutes at 37uC. The contents of the

reaction including the remainder of the fat pad and supernatant

were passed though a 40 mm cell strainer (BD Falcon, Franklin

Lakes, NJ) and gently ground using the plunger of a syringe. Both

the syringe and filter were washed using more supplemented

media. Following centrifugation (2000 RPM, 5 minutes), red cells

were lysed using 1X Pharmlyse (BD Biosciences, San Jose, CA).

Single cell suspensions were then stained to indentify macrophages

(F4/80) and immature myeloid cells (F4/80, Gr1, Mac1). B-cells

were identified based on B220 expression and T-cells were

identified based on CD8 expression. All antibodies were purchased

from BD Biosciences.

StatisticsAll data are presented as means 6 SD. Student’s t-tests were

used to test differences between groups at each time point. All

statistical tests were performed using SPSS software (IBM, Somers,

NY) assuming a significance level of P,0.05.

Results

High Fat Feeding Rapidly Induces the Obese PhenotypeTwo days of HFD resulted in a +4% increase in body weight

(n.s.) and a +28% increase in epididymal fat pad mass compared to

RD (P,0.01; Table 1). One week on the HFD regimen increased

body mass by +9% (P,0.05), and increased epididymal fat mass

by +118% (P,0.01) as compared to RD. After six weeks, HF

animals were +23% heavier (P,0.01), and epididymal fat mass

was +300% greater than that of RD mice (P,0.01). After 6 w of

HFD, total abdominal adiposity was +411% greater than that

measured in RD (P,0.01; Fig. 1).

Both visceral and subcutaneous adiposity increased over the six

week HFD protocol, with the greatest increases being measured in

the visceral compartment. As compared with a ratio of 4269.9%

visceral fat to total adiposity in RD, this ratio increased

significantly in HF mice to 6463.9%, representing a marked shift

of adiposity to the visceral compartment (P,0.01; Fig. 1).

Leukocyte Infiltration into Adipose Tissue IncreasesFollowing Shift to High Fat Diet

The onset of obesity engenders an influx of leukocytes into

adipose tissue, which are central to adipose remodeling and the

inflammatory cascade which parallels obesity. As measured at

3 W, HFD led to a significant invasion of leukocytes into

epidydimal fat, as reflected by a +80% higher population of

macrophages in HF mice as compared to control (P,0.01). High

fat feeding also increased the presence of immature myeloid cells

by +20% (P,0.05), while CD8 T-cells increased +60% (P,0.01).

Interestingly, the proportion of B-cells had not increased by 3W of

HFD, as HF had 26% fewer B-cells than LF mice (n.s.).

Six Weeks of HFD Increases Marrow AdiposityFollowing six weeks of HFD, the adipose tissue fraction of BM

increased by +363% compared to RD (P,0.01). Similarly, the

average adipocyte area, measuring 6366328 mm2 in RD mice,

increased +72% in HF to 10936672 mm2 P,0.01;Fig. 2). At this

same time point, triglycerides measured in the bone marrow

increased by +48% in HF mice as compared to RD controls

(P,0.01).

High Fat Diet Disrupts Hematopoietic Lineage AllocationLeukocytes isolated from the peripheral blood and bone

marrow were used for analysis of B-cells, T-cells and myeloid

lineages (Fig. 3). HFD had no impact on the level of circulating

leukocytes in the peripheral blood at any time point studied here,

and preliminary data indicates that enhanced extramedullary

hematopoiesis may have played a role in maintaining these

circulating populations (data not shown). In contrast, the

proportions of leukocytes in the bone marrow were significantly

compromised by the high fat diet regimen. After 2 d, HF animals

had 228% fewer T-cells than RD (P,0.05), and 25% fewer

myeloid cells (P = 0.07) in the bone marrow, with no differences

seen between HF and RD B-cell populations at this time point.

After 1 w, however, the impact of HFD on leukocyte populations

had reversed, with mice fed a high fat diet displaying decreased B-

cells (210%, P,0.05), but no differences measured between HF

and RD in either myeloid or T-cells.

By six weeks, the bone marrow displayed an ‘‘aged’’ phenotype

in HF animals [22] with the proportion of T-cells falling by

231%, and B-cells down by 225% as compared to RD (P,0.01),

while the myeloid cell fraction rose by +16% (P,0.01; Fig. 3).

These data emphasize the vulnerability of hematopoietic lineages

to HFD even within a very short time period, and that this

disruption persists over time.

In order to understand if leukocyte populations were truly

changing, or merely changing with respect to one another, we also

extrapolated to the total population sizes by multiplying the

measured proportion by the total number of cells harvested for

flow cytometry. Total cellularity was not different between the

groups at any time point, however there was a trend of increased

cell content at 6 W in the HF animals (+13%, P = 0.08). The total

number of T-cells tracked very well with their phenotypic

proportions being 232% and 230% lower in HF than RD

animals at 2D and 6W (P,0.05), and not different from RD at the

1W time point (+2%, n.s.). Myeloid population sizes also closely

matched the reported proportions, with 29% fewer myeloid cells

in HF animals after 2D (P = 0.08), a non-significant +13% increase

at 1W, and a +16% increase after 6W of HFD (P,0.01).

Interestingly B-cell population sizes bucked the trend of their

proportional counterparts at the 1W time point. Whereas

proportionally there were 210% fewer B-cells in HF animals at

1W, in estimated total numbers this decrease was only 24% (n.s.).

Both the 2D and 6W time points did correspond to the

proportional data, as a deficit of total B-cells emerged at the 6W

time point (225%, P,0.01)(Fig. 4).

Transient Hematopoietic Response to HFDTo determine if the HFD mediated disruption of leukocyte

allocations were paralleled by changes to the primitive hemato-

poietic system, HSCs and their earliest progeny were isolated from

the bone marrow. No HFD mediated differences were observed in

the primitive SP-KLS population at any time point, perhaps an

indication of these cells’ quiescence, especially over a short term of

exposure to HFD.

While HSCs were not significantly impacted by diet, HFD did

influence the more differentiated hematopoietic progenitor cells

(KLS). After 2 d, this population was non-significantly elevated in

HF by +15%, while within one week the KLS cell fraction

increased by +16% relative to RD (P,0.01; Fig. 5). This increase

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in hematopoietic progenitors may be indicative of an early strategy

to restore and rebalance leukocyte differentiation as disrupted by

HFD. However, this reparative response measured in the HF mice

was transient, as the +7% greater number of progenitor cells

measured at 6 weeks was not significantly different from that seen

in RD.

HFD Developmentally Limits Bone MarrowLymphopoiesis

To examine the origins of the lymphopenic phenotype, RT-

PCR was performed on whole bone marrow extracted from

femurs at 2D, 1W, and 6W (Fig. 6). After 2 d of HFD, the

expressions of Il-7 and Ebf-1, critical to early B-cell development,

were down 220% (P = 0.08) and 211% (P = 0.06), while Pax-5

expression, a marker indicating an intermediate stage of develop-

ment, showed only a slight trend in reduction relative to RD

(29%; p = 0.18). At 1 w, the expression of all three genes was

suppressed in the HF mice, with Il-7 decreased by -19%, Ebf-1

reduced by 220%, and Pax-5 down by 216% (P,0.01 in each)

relative to RD. These relative reductions persisted at 6 w, with the

expression of Il-7, Ebf-1, and Pax-5 down-regulated by 223%,

229%, and 234%, respectively (P,0.01). Runx1 and Crebbp

expression were decreased after 6 w in HF by 217% and 224%,

as compared to RD (P,0.05). The expressions of CXCL-12 and

Angiopoietin-1 were not changed at either 2D or 6W.

Discussion

Obesity fosters a host of chronic, systemic health complications

which conspire towards a marked decrease in life expectancy [23].

That obesity also increases the risk of infection and illness [24,25],

Figure 1. High Fat Diet Induces an Obese Phenotype. (a,b) At 6 weeks HF animals have expanded total abdominal adiposity with a prominentshift in the location of this adiposity from primarily subcutaneous to the visceral compartment. (c) The expansion and relocation of adiposity isillustrated in 3D reconstructions of abdominal mCT images which have been thresholded for adiposity (n = 9). In these images gray representssubcutaneous fat and pink represents visceral fat. *P,0.05 vs. RD.doi:10.1371/journal.pone.0090639.g001

Table 1. High Fat diet leads to a significant expansion ofepidydimal fat after 1 and 6 weeks compared to RD controls,demonstrating the rapid accretion of adiposity in mice fedthis diet.

Epididymal Fat Weight (g) RD HF

2 Days 0.3060.06 0.3860.06*

1 Week 0.3060.06 0.6560.18*

6 Week 0.3460.09 1.3660.46*

*P,0.05 vs. RD.doi:10.1371/journal.pone.0090639.t001

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implicates that elevated adiposity ultimately impairs immunity. In

the work presented here, we examined the possibility that a high

fat diet could rapidly disrupt hematopoiesis even during the

earliest stages of the initiation of obesity.

It is well recognized that obese mice present with diminished

immune cell function [16,26]. In addition to the impairment of

leukocyte function per se, obesity weakens the health of hemato-

poietic tissues and the maintenance of appropriate population sizes

of leukocytes. Indeed, Yang et al. showed that frank obesity as

caused by 11 months of a HFD was correlated to severely

compromised lymphoid lineages in the bone marrow, leading to

thymic involution and defective T-cell production [27]. Addition-

ally, obesity as caused by seven months of a HFD (45% kcal from

fat) was associated with B lymphopenia amongst both developing

(BM) and circulating cells, as well as significant trabecular bone

loss, an indication of deleterious trends in the BM niche [14].

Interestingly, only circulating T-cells, and not those in the bone

marrow, were disrupted by HFD, suggesting that T-cell popula-

tions are less indicative of the health of the bone marrow than B

lymphocytes; at least when impacted by changes in diet. The

central role that BM plays in the genesis and development of

leukocytes led us to hypothesize that any impact of a high fat diet -

as a prequel to obesity - on immune populations might be initiated

by early disruption of the BM environment and thus the cells that

reside in it. In this study, we set out to trace the origins of such

lymphopenic phenotypes both in terms of initial occurrence and

their relationship to changes in the niche.

The impact of high fat diet on lymphopoiesis was seen as early

as two days, as evidenced by a subtle decline in BM T-cell

populations. T-cells recovered to normal population levels by 1w,

but regressed again to levels lower than RD controls at 6w.

Whereas previous reports have linked long term T lymphopenia in

obesity to developmental issues in the thymus [27], the early

decline and then recovery of BM T-cells observed here suggests

that, at least initially, T-cell production is not impaired by obesity

per se, but rather these populations may be recruited elsewhere in

response to a systemic increase in the adipose burden initiated by

the high fat diet. T-cell populations have been suggested to

precede macrophage infiltration of visceral adipose tissue, helping

to initiate the inflammatory cascade in rapidly expanding adipose

depots [28]. That these fluctuations happen so quickly may imply

an insult caused by diet as much as by the change in the adipose

phenotype, and in reality, they may both contribute to the change

in T-cell levels. Indeed, as evidenced by data from the 3W

protocol, the obese mice showed a significant infiltration of CD8

T-cells into the adipose tissue. Additionally, populations of

macrophages and immature myeloid cells increased significantly

in the fat pads. Similar data has been shown previously elsewhere,

as already indicated, however in this case these data are used to

illustrate the rapid systemic effects of high fat diet. Specifically,

short durations of high fat diet strongly influence inflammation

Figure 2. Six Weeks of HFD Rapidly Remodels the Bone Marrow Towards Adiposity. (a,b,d) The percentage of BM space taken up byadipose tissue, as assessed by histology, increased by almost 4 fold in HFD animals compared to control, an increase at least partially due toincreasing adipocyte size (n = 6). (c) This leads to an increase in BM triglyceride storage (n = 10). The inclusion of adipocytes in the BM is significantbecause of the deleterious paracrine and inflammatory consequences adipocytes are thought to present to hematopoiesis. Additionally, BM adiposityin general represents a decline in BM quality and health, as is demonstrated by increased BM adiposity in osteoporosis and anorexia nervosa. *P,0.05vs. RD.doi:10.1371/journal.pone.0090639.g002

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and leukocyte populations and trafficking in diverse anatomical

sites, even in the absence of an impact on circulating populations.

Unlike T-cells, which exit the bone marrow at a relatively early

stage of development to mature in the thymus, B-cells remain in

the BM niche further into their maturation. This prolonged

exposure of the B-cell progenitor pool to the disrupted regulatory

environment of the bone marrow invariably would make B-cell

development more susceptible to the changes in the BM

phenotype. For this reason, particular emphasis was placed on

understanding the influence of short term HFD and obesity on the

B-cell lineage.

These studies indicate that a high fat diet rapidly catalyzes a

decline in B-cell populations, the origins of which appear to begin

at the earliest stage of B-cell commitment. The first phenotypic

indication of a B-cell defect occurred at 1w, degenerating further

by 6w at which point total numbers of B-cells were also reduced.

Additionally, despite the relative youth of these animals (13 weeks

at time of sacrifice), the overall bone marrow phenotype displayed

classic signs of age-related dysfunction, characterized by myelo-

proliferation and diminished lymphopoiesis [22]. This rapid

disruption of the bone marrow phenotype demonstrates both

local and systemic consequences of a high fat diet, and its

associated adiposity, and its capacity to harm leukogenesis.

Supporting a conclusion that the high fat diet disrupts fate

selection in the earliest stage of HSC progenitors, RT-PCR was

used to show changes in a range of genes that indicated that the

depletion of B-cells could be traced back along the differentiation

pathway to even the earliest points in commitment to the lineage.

For example, Ebf-1 is recognized as an important regulatory and

survival factor in the earliest stages of B lymphopoiesis [29], and is

required for lineage commitment as well as the successful

transition from the pro- to pre-B stage of development [30]. The

initiation of Ebf-1 expression has been recently linked to a

functional switch within the CLP compartment associated with B-

cell lineage restriction. This switch is dependent on Il-7, a cytokine

produced by hematopoietic stroma [8]. Our data demonstrated

strong trends of reduced Il-7 and Ebf-1 expression after only 2d of

HFD, prior to a significant reduction of Pax-5 expression or any

reduction of B220+ B-cells. Following 1w of HFD, Il-7, Ebf-1 and

Pax-5 were all significantly down regulated, correlating well with

an emerging depletion of phenotypic B-cells. However, despite the

reduction in the phenotypic B-cell proportions, total numbers of B-

cells were not reduced until the 6W time point. This fact combined

with the early down regulation of B-cell differentiation factors

points to the developmental nature of the B-cell depletion,

originating in diminished maturation of primitive B-cells. Contin-

ued exposure to a high fat diet through six weeks led to further

suppression of B lymphopoiesis at each stage examined, from

lineage restriction (Il-7 and Ebf-1), through intermediate develop-

mental stages (Pax-5) including a robust suppression of phenotypic

B-cells (B220+), ultimately resulting in the aforementioned

reduction in population size. Therefore, we conclude that a high

fat diet induces B lymphopenia by developmentally limiting the

production of B-cells.

Figure 3. High Fat Diet Initiates an Aged Leukocyte Phenotype in the Bone Marrow. (a) A two-color flow cytometric method was used toisolate leukocyte populations of the lymphocyte (B and T-cells) and myeloid lineages, with the unstained control displayed in the left panel. (b–d) Attwo days a T-lymphopenia was present. At one week of HFD B-cell populations were diminished in the HF group, while T-cell populations were notsignificantly affected. After six weeks bone marrow lymphocyte populations were depressed and the myeloid lineage proliferated in response to HFD.The suppression of lymphocytes accompanied by myeloid expansion is a classic phenotype of hematopoietic aging. These changes may lead todefects in adaptive immunity. Each time point was drawn from distinct cohorts of animals, as such, comparisons can only be made within a timepoint due to variability in processing. *P,0.05 vs. RD.doi:10.1371/journal.pone.0090639.g003

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The early down regulation of Il-7 and Ebf-1, at the head of the

B lineage, signifies that the change to a high fat diet disrupts

leukogenesis at early stages of commitment, in this case by

depriving progenitors of critical environmentally derived differen-

tiation cues. We hypothesize that it is the reduction in Il-7

expression which initiates lymphopenia by restricting lineage

commitment, indicating that changes to the BM niche are

responsible for the hematopoietic disruption observed in these

studies. The ongoing reduction of Il-7 may further amplify the

lymphopenic phenotype by harming the survival of Il-7r+ cells,

many of which are committed B progenitors [8]. Recent work has

implicated IL-7 production by osteoblasts as being critical to B-

lymphopoiesis [31]. Osteoblasts have long been known to play

important roles in the hematopoietic niche, particularly in

regulating B-cells [32,33]. These data suggest an influence of

obesity on osteoblasts, however more work is needed to clarify the

mechanisms through which systemic or local adiposity might

diminish osteoblastic IL-7 production. Interestingly, the expres-

sions of CXCL-12 and Angiopoietin-1 were not reduced by the

onset of obesity. These genes encode proteins which, similar to IL-

7, are involved in the maintenance of hematopoietic cells in niches

comprised of single or few cell clusters. The fact that the

expression of these genes was unaffected implies that IL-7+osteoblasts, and not other niche cells and ligands, were impacted

by obesity in a targeted, rather than generalized manner.

The susceptibility of Il-7 expression in the bone marrow, and by

extension B lymphopoiesis to a high fat diet has not previously

been reported, and illustrates the delicate balance within the

hematopoietic niche and the populations that depend on it. These

data reflect the speed with which this change to a high fat diet and

concomitant obesity causes functional and phenotypic changes in

the niche, and the long-term consequences to the immune system

that these initiate. This conclusion is supported by the marked

changes in the composition of the marrow niche, driving towards

3-fold increases in adipose burden in the marrow cavity after only

6 weeks of HFD. BM adiposity is widely thought to hamper

hematopoiesis through direct paracrine and inflammatory insults

[11]. What is interesting is that BM adiposity is not unique to

obesity, but rather is a phenotype present in various states of

impaired health, such as osteoporosis, aging, anorexia, and models

of irradiation induced bone marrow failure [34,35]. This may

imply that BM adiposity, in addition to a causative role in the

development of obese hematopoietic defects, may also be a

concomitant result and signal of diminished BM quality.

Injury to the hematopoietic system is often paralleled by an

increase in hematopoietic progenitor (KLS cell) proliferation, a

response interpreted as a central reparative strategy to restore

balanced hematopoiesis [36]. The scale of this reparative response,

and the exact populations involved are suspected to vary with the

nature and severity of injury [3]. We believe these data provide

further evidence of the consequences of a dietary stressor to

rapidly disrupt hematopoietic populations, an imbalance which in

turn signals HSCs to differentiate towards hematopoietic progen-

itors as a step towards restoring the leukocyte reservoir.

Similar to the significant elevation of KLS populations within

one week of the introduction of a high fat diet, infection with

vaccinia virus has been shown to lead to a rapid (1 day) increase in

bone marrow KLS cells, a response which subsides when leukocyte

progenitors show evidence of normalizing to healthy levels [37].

However, the continued restriction of Il-7 expression indicates that

Figure 4. High Fat Diet Alters the Total Population Sizes of BM Leukocytes. (a) Total BM cells harvested for flow cytometry were notdifferent between HF and RD animals at any time point, but a trend of increase was observed at 6W. (b) The total number of B-cells was not changedat 2D or 1W contrary to a decline in the phenotypic population at 1W. However, B-cells were reduced at 6W, despite a slight increase in totalcellularity, demonstrating the degree of population reduction. (c,d) Total population sizes of both T-cells and Myeloid lineages tracked well with thephenotypic population proportions. *P,0.05 vs. RD.doi:10.1371/journal.pone.0090639.g004

High Fat Diet Rapidly Suppresses B Lymphopoiesis

PLOS ONE | www.plosone.org 7 March 2014 | Volume 9 | Issue 3 | e90639

such attempts at repair might be undermined a priori, unless and

until the aggravating factor itself is removed. Indeed, while KLS

cells in HFD displayed a significant elevation in response to diet-

induced damage at one week, by six weeks this progenitor pool

appears unable to sustain these elevated levels and returned to

those levels measured in RD. While an extrapolation, we interpret

the retreat of hematopoietic progenitor populations in the high fat

diet group - despite evidence of continued leukocyte dysregulation

- as an indication of a systemic collapse of the hematopoietic

reparative response in spite of persistent dietary and adipose

challenges. Indeed, our data also indicate a loss of transcriptional

control in more primitive HSCs, as seen in the lowered expression

of Runx1 and Crebbp at 6w. Both of these genes are transcription

factors which play crucial roles in hematopoietic development and

quiescence, and it has been postulated that their expression is

antagonistic to myelopoiesis [38,39,40]. The decrease of expres-

sion in these genes implies a loss of HSC regulation and, perhaps

ultimately, loss of function due to both a high fat diet and the

obese phenotype. As both Runx1 and Crebbp have been

associated with HSC regulation through intrinsic and niche

mediated mechanisms, it is not clear at this stage whether this is

another consequence of high fat diet initiated damage to the niche,

or of direct impact on the HSCs [41,42]. What is clear from these

data is that continued exposure to a high fat diet has the potential

to markedly disrupt hematopoiesis, from the earliest stages of

primitive HSC lineage selection to the ultimate production and

function of leukocytes.

Conclusions

These data demonstrate that introduction to a high fat diet

catalyzes a rapid disruption of the BM niche, the consequences of

which include dysregulation of B-cell populations. Further, our

data suggest that defects in lymphogenesis are the result of

impairments to niche function early in the development process,

resulting in diminished cellular differentiation. It is currently

unclear if these processes are mediated primarily by obesity, or if

the increase in adiposity and some combination of dietary factors

Figure 5. Hematopoietic Progenitors Rapidly Respond to High Fat Diet Mediated Damage. (a) A representative gating scheme to isolateKLS and SP-KLS cells is shown. HSCs (SP-KLS) were assessed to determine the hematopoietic response to obesity driven leukocyte mis-allocation butno differences were found at any time point studied. (b) Hematopoietic progenitors rapidly respond to obesity mediated damage displaying an earlyresponse to HFD with HF animals having elevated KLS populations at 1 week. By 6 weeks this response was no longer evident in HF animals. *P,0.05vs. RD.doi:10.1371/journal.pone.0090639.g005

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PLOS ONE | www.plosone.org 8 March 2014 | Volume 9 | Issue 3 | e90639

are causative. Although a high fat diet also appears to cause

dysregulation to more primitive HSCs, it is unclear if this damage

is intrinsic, or niche mediated, and to what extent this damage is

already manifesting in the development of leukocyte populations.

Acknowledgments

The authors wish to thank Andrea Trinward and Steven Tommasini for

assistance in tissue processing, and Alyssa Tuthill for administrative

support.

Author Contributions

Conceived and designed the experiments: BJA CTR. Performed the

experiments: BJA DEG GP MEC. Analyzed the data: BJA DEG GP MEC.

Wrote the paper: BJA CTR.

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